The goal of this project is to elucidate the molecular mechanisms underlying the functions of apolipoprotein (apo) A-I in reverse cholesterol transport (RCT). Apo A-I is the major protein of plasma high density lipoprotein (HtDL) and the functions of this molecule underlie the anti-atherogenic properties of this lipoprotein. Specific Aim 1 is to define the properties of apo A-I amphipathic alpha-helices required for optimal solubilization of phospholipid (PL), and high affinity binding to lipid and lipoprotein particles of different sizes. A range of engineered apo A-I molecules and synthetic peptides with altered alpha-helix properties will be studied using physical-biochemical methods to establish the quantitative parameters that control lipid binding. Specific Aim 2 is to determine the features of the apo A-I molecule that are critical for it to form nascent (pre-beta) HDL particles by binding PL and cholesterol molecules delivered from a cell plasma membrane via the ATP-binding cassette transporter A1 (ABCA1). The kinetics of PL and cholesterol efflux from ABCA1-expressing fibroblasts and macrophages growing in culture to the engineered apo A-I molecules will be measured; the structures of the nascent HDL particles created in the extracellular medium will also be determined. The cholesterol transport properties of these particles will be explored in collaboration with Project 1. Specific Aim 3 is to define the lipidation of apo A-I and the properties of alpha-helices required for binding to scavenger receptor class B, type I (SR-BI) so that the subsequent selective uptake of HDL lipids into cells is optimal. Different HDL particles containing engineered apo A-I molecules will be incubated with SR-BI-expressing cells to examine how HDL structure modulates lipid transfer to the cells. In collaboration with Project 3, variant apo A-I molecules with altered functionality will be expressed in mice and the effects on HDL metabolism, RCT and atherosclerosis will be determined. The results of this project wilt provide greater understanding of the mechanisms by which apo A-I and HDL protect against premature coronary artery disease.